A polyclonal antibody of colp6-2 protein of marnifia vesfa, application and preparation method thereof

Abstract

The application discloses a kind of Marninif basket fungus colp6-2 protein polyclonal antibody and preparation method, with the amino acid sequence shown in SEQ ID NO.1 as immunogen, first obtain the recombinant protein of the amino acid sequence;Meanwhile, inactivated Marninif basket fungus is used to immunize domestic rabbit, and antiserum is obtained by blood taking;Then the polyclonal antibody of Marninif basket fungus colp6-2 protein is obtained by using recombinant protein to purify antiserum, the polyclonal antibody can both antigen identify Marninif basket fungus;And can recognize recombinant protein antigen, specificity is strong, and detection sensitivity is high.The application also discloses a kit for detecting Marninif basket fungus, and the biotin-labeled Marninif basket fungus colp6-2 protein polyclonal antibody is used to detect Marninif antigen in sample, and the detection specificity is strong, and the sensitivity is high.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a polyclonal antibody against the colp6-2 protein of *Basilella marneffei*, its application, and its preparation method. Background Technology

[0002] *Penicillium marneffei* (PM), also known as *Basilella marneffei*, is a dimorphic fungus that causes marneffei fungal infection (PSM), a rare systemic disease caused by a deep fungal infection. *Basilella marneffei* infection is most common in southern China and Southeast Asia. While it can occur in healthy individuals, it is more prevalent in immunocompromised or immunosuppressed individuals. Studies in Vietnam have shown that PSM ranks third among opportunistic infections in AIDS patients. With the increasing number of HIV infections, reports of *Penicillium marneffei* infection are also rising annually.

[0003] Existing literature reports that *Basilaria marneffei* glycoproteins and *Aspergillus* glycoproteins have the same or similar antigenic determinants, making cross-reactivity in immunological detection easy. Furthermore, glycoproteins are easily cleared by phagocytes after binding with antibodies. However, a rapid, sensitive, and specific method for detecting *Basilaria marneffei* antigens has not yet been developed. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a polyclonal antibody against the colp6-2 protein of *Basilella marneffei*, its application, and its preparation method.

[0005] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0006] A method for preparing a polyclonal antibody against the colp6-2 protein of *Basilella marneffei* includes the following steps:

[0007] Step S1: The antigen sequence clp6-2 is determined by sequence alignment. The amino acid sequence of the antigen sequence clp6-2 is shown in SEQ ID NO.1, and the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO.2.

[0008] Step S2: The nucleotide sequence shown in SEQ ID NO.2 is transferred into the expression vector pET28a to construct a recombinant expression vector. The recombinant expression vector is then transformed into Escherichia coli, and the plasmid is extracted.

[0009] Step S3: Transform the plasmid of the recombinant expression vector into Escherichia coli to obtain a recombinant expression strain, express the target protein in the recombinant expression strain, and purify to obtain the recombinant protein;

[0010] Step S4: Immunize rabbits with inactivated *Basilus marneffei*, collect blood, centrifuge to obtain antiserum, and purify the antiserum with the recombinant protein obtained in step S3 to obtain a polyclonal antibody against *Basilus marneffei* colp6-2 protein.

[0011] In step S2, the method for constructing the recombinant expression vector is as follows: using cDNA of *Basilella marneffei* as a template, PCR amplification is performed to obtain the nucleotide sequence shown in SEQ ID NO.2, and the nucleotide sequence is transferred into the restriction site between BamHI and XhoI of the expression vector pET28a to obtain the recombinant expression vector; preferably, the upstream primer sequence used for the PCR amplification is shown in SEQ ID NO.3, and the downstream primer sequence is shown in SEQ ID NO.4.

[0012] In step S2, the recombinant expression vector is transformed into Escherichia coli BL21(DE3), and E. coli transformed into the recombinant expression vector are screened using a plate medium containing kanamycin, and then the plasmid is extracted.

[0013] In step S3, the plasmid of the recombinant expression vector is transformed into Escherichia coli BL21(DE3), and the recombinant expression strain is screened using a plate medium containing ampicillin.

[0014] In step S3, the recombinant expression strain is inoculated into a liquid culture medium containing ampicillin to obtain a bacterial culture, IPTG inducer is added to induce the expression of the target protein, and then the bacterial cells are collected by centrifugation, the bacterial cells are sonicated and purified using a Ni-NTA column to obtain the recombinant protein; preferably, the final concentration of IPTG inducer in the bacterial culture is 0.5 mM, the induction temperature is 30℃, and the culture is shaken for 3 h to induce the expression of the target protein.

[0015] In step S4, the method for purifying antiserum with the recombinant protein obtained in step S3 to obtain polyclonal antibodies against the *Bambusa marneffei* colp6-2 protein is as follows: the recombinant protein obtained in step S3 is prepared into an antigen affinity column, a mixture of antiserum and buffer solution is loaded onto the column, and then eluted with 0.1M citric acid solution. The eluent is collected, concentrated, and then the polyclonal antibody against the *Bambusa marneffei* colp6-2 protein is obtained.

[0016] This invention protects the polyclonal antibody against the *Bambusa marneffei* colp6-2 protein prepared according to the above method.

[0017] The present invention also protects the application of the polyclonal antibody against the *Basilella marneffei* colp6-2 protein, for any one of the following (a1) to (a6):

[0018] (a1) Identification of *Basilella marneffei*;

[0019] (a2) Prepare a kit for identifying *Basilella marneffei*;

[0020] (a3) Detect whether the pathogenic microorganism to be tested is *Basilella marneffei*;

[0021] (a4) Prepare a kit for detecting whether the pathogenic microorganism to be tested is *Basilella marneffei*;

[0022] (a5) Detect whether the sample to be tested contains *Basilella marneffei*;

[0023] (a6) Prepare a kit for detecting whether a sample contains *Bambusa marneffei*.

[0024] This invention also protects a kit containing a polyclonal antibody against the *Cladosporium marneffei* colp6-2 protein, wherein the application of the kit is any one of the following (b1) to (b3):

[0025] (b1) Identification of *Basilella marneffei*;

[0026] (b2) To determine whether the pathogenic microorganism to be tested is *Basilella marneffei*;

[0027] (b3) Detect whether the sample to be tested contains *Bambusa marneffei*.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The present invention first prepares a recombinant protein with a partial sequence of the colp6-2 protein of *Bacillus marneffei*; at the same time, rabbits are immunized with inactivated *Bacillus marneffei* and blood is collected to obtain antiserum; then the antiserum is purified with the recombinant protein to obtain a polyclonal antibody against the colp6-2 protein of *Bacillus marneffei*. This polyclonal antibody can both recognize *Bacillus marneffei* antigen and recognize the recombinant protein antigen, and has high specificity and high detection sensitivity.

[0030] (2) The present invention also discloses a kit for detecting Bassula marneffei, which uses a biotin-labeled polyclonal antibody against the colp6-2 protein of Bassula marneffei to detect the marneffei antigen in the sample, with high specificity and high sensitivity.

[0031] Instruction manual illustrations

[0032] Figure 1 This is a diagram showing the SDS-PAGE electrophoresis detection results of Embodiment 1 of the present invention.

[0033] exist Figure 1Lane 1 - Pre-induction bacterial culture, Lane 2 - Post-induction bacterial culture, Lane 3 - Marker, Lane 4 - Broken bacterial cell precipitate, Lane 5 - Supernatant after broken bacterial cells, Lane 6 - Effluent from the column, Lane 7 - 5% imidazole for eluting impurities, Lane 8 - 30% imidazole for eluting proteins, Lane 9 - 100% imidazole for eluting proteins. Detailed Implementation

[0034] The following detailed description is provided in conjunction with specific embodiments, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise specified, all raw materials used in the embodiments are commercially available.

[0035] Example 1: Preparation of polyclonal antibody against *Bassula marneffei* colp6-2 protein

[0036] Step S1: The antigen sequence clp6-2 is determined by sequence alignment. The amino acid sequence of the antigen sequence clp6-2 is shown in SEQ ID NO.1, and the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO.2.

[0037] Step S2: The nucleotide sequence shown in SEQ ID NO.2 is transformed into the expression vector pET28a to construct a recombinant expression vector. The recombinant expression vector is then transformed into E. coli, and the plasmid is extracted. The specific experimental steps are as follows:

[0038] Step S21: Using the cDNA of *Basilella marneffei* as a template, PCR amplification was performed, and the amplified target gene fragment was recovered to obtain the nucleotide sequence shown in SEQ ID NO.2.

[0039] The upstream primer sequence used for PCR amplification is (SEQ ID NO.3): 5'- GGATCC GGTAGCAGCTATGTTGATTATCA-3', the underlined part is the recognition sequence of the BamHI restriction site; the downstream primer sequence is (SEQ ID NO.4): 5'- CTCGAG TTAGTTAATGCTGGTTGCACCG-3', the underlined part is the recognition sequence of the restriction enzyme site XhoI;

[0040] The PCR amplification reaction system is as follows: 1 μL upstream primer, 1 μL downstream primer, 1 μL cDNA template (20-50 ng), 5 μL 10×pfu buffer, 1 μL pfu (5 U), and deionized water to make up to 50 μL.

[0041] The PCR amplification reaction program was as follows: 95℃ for 5 min; 95℃ for 15 s, 55℃ for 2 Kb / min, 72℃ for 1 min, repeated for 25 cycles; 72℃ for 10 min; end the reaction.

[0042] Step S22: The target gene fragment and the expression vector pET28a are digested with enzymes respectively;

[0043] The enzyme digestion reaction system is as follows: 1-2 μg of expression vector pET28a / target gene fragment, 5 μL of 10×cutsmart buffer, 1 μL of restriction endonuclease BamHI, 1 μL of restriction endonuclease XhoI, and deionized water to a final volume of 50 μL.

[0044] The enzyme digestion reaction conditions are: incubation at 37℃ for 1-2 hours.

[0045] Step S23: The enzyme-digested expression vector pET28a is ligated with the target gene fragment to obtain the recombinant expression vector;

[0046] The ligation system consisted of: expression vector pET28a 50 ng, target gene fragment 150 ng, 10×T4 DNA Ligase buffer 2 μL, T4 DNA Ligase 1 μL, and deionized water to a final volume of 20 μL.

[0047] The ligation reaction conditions were: incubation at 16℃ for 0.5–1 h.

[0048] Step S24: Add the recombinant expression vector to a centrifuge tube containing TOP10 competent cells (25 ng DNA is required for every 50 μL of competent cells). Place the centrifuge tube in a circulating water bath at 42°C for 90 seconds to heat shock. Quickly transfer the centrifuge tube to an ice bath to cool the cells for 1–2 minutes. Add 200 μL of SOC liquid medium to each tube and heat the medium to 37°C in a water bath. Then transfer the centrifuge tube to a shaker at 37°C and incubate at 220 rpm for 45 minutes to revive the cells and express the resistance marker gene encoded by the plasmid. Transfer the competent cells to LB agar plates containing kanamycin and incubate at 37°C for 12–16 hours. Pick colonies for PCR verification. Then inoculate the colonies that have been verified to have transformed with the target gene fragment into liquid medium and incubate until a certain OD value is reached before extracting the plasmid.

[0049] Step S3: The plasmid of the recombinant expression vector is transformed into *E. coli* to obtain a recombinant expression strain. The target protein is expressed in the recombinant expression strain, and the recombinant protein is purified. The specific experimental steps are as follows:

[0050] Step S31, competent cell transformation and positive clone screening: Rosetta (DE3) competent cells were removed from the ultra-low temperature and thawed on ice; plasmid (5 μg) was added, and the mixture was gently pipetted and aspirated to mix thoroughly, and placed on ice for 30 min; the cells were then heat-shocked at 42°C for 90 s and placed on ice for 1 min; 800 μL of LB liquid medium preheated to 37°C was added, and the cells were incubated at 37°C and 158 rpm for 50 min; the cells were centrifuged at 6000 rpm for 4 min, and a portion of the supernatant (800 μL volume) was removed. The remaining bacterial culture was mixed and spread onto ampicillin agar plates; the plates were inverted and incubated at 37°C for 12–16 h until colonies (positive clones) appeared;

[0051] Step S32, Protein Expression and Bacterial Disruption Detection: Positive clone colonies were inoculated into 100 mL of LB liquid medium (containing ampicillin) and cultured overnight with shaking. 100 mL of bacterial culture was inoculated into 2000 mL of LB liquid medium and cultured at 37°C until the OD600 reached approximately 0.6. The culture temperature was then lowered to 30°C. IPTG inducer was added to a final concentration of 0.5 mM, and the culture was continued at 30°C with shaking for 3 hours. The cells were collected by centrifugation at 8000 rpm for 3 minutes and resuspended in 50 mL of pre-chilled NTA-0 buffer, then incubated on ice for 30 minutes. The cells were then sonicated using the following parameters: power 200 W, 3 seconds on, 4 seconds off, 99 cycles. The cells were centrifuged at 16000 rpm at 4°C for 50 minutes, and the supernatant and precipitate were collected. A small amount of the supernatant and precipitate was analyzed by SDS-PAGE, and the remaining supernatant and precipitate were stored at 4°C for later use.

[0052] Step S33, recombinant protein purification: Filter the supernatant from the lysed bacterial cells using a 0.22 μm filter; prepare a Ni-NTA column, load the supernatant protein solution at a flow rate of 1 mL / min, wash the column with NTA-0 buffer (pH 8.0) until the eluent is free of protein (G250 detection solution does not change color); elute with 20 mM, 60 mM, 200 mM, and 500 mM imidazole respectively, collect the eluent fractions until the G250 detection solution does not change color; wash the column material with 3 column volumes of deionized water, and seal the column with 20% ethanol; perform SDS-PAGE electrophoresis on the collected eluent.

[0053] The SDS-PAGE electrophoresis results in this step are as follows: Figure 1 As shown, from Figure 1 As can be seen, the protein eluted by 30% imidazole in lane 8 has a distinct band with a size of approximately 25 kDa, which is also the expected size of the fusion protein in this experiment, indicating that the protein was successfully purified.

[0054] Step S4: Immunize rabbits with inactivated *Basilus marneffei*, collect blood, centrifuge to obtain antiserum, and purify the antiserum with the recombinant protein obtained in step S3 to obtain a polyclonal antibody against *Basilus marneffei* colp6-2 protein. Specific experimental steps are as follows:

[0055] Step S41, Immunization: Immunize rabbits with inactivated Bacillus marneffei emulsion for a total of 6 immunizations, with an interval of 2 weeks between each immunization. Take a small amount of antiserum to test the titer. After the titer reaches the target, take blood, incubate overnight at 4°C, and centrifuge at 4000 r / min for 10 min to obtain antiserum.

[0056] Step S42, Preparation of antigen affinity column: The recombinant protein obtained in step S3 is used to prepare an antigen affinity column using conventional techniques;

[0057] Step S43, Purify antiserum: Wash and equilibrate the antigen affinity column with 10 column volumes of PBS (or TBS, the same below). After centrifuging the antiserum at high speed, mix the supernatant with an equal volume of 2×PBS buffer, adjust the pH and ion concentration, and slowly add it to the antigen affinity column; then wash with more than 10 column volumes of PBS until no protein is detected in the effluent; add 2 column volumes of 0.1M citric acid (pH 2.7), clamp the effluent tube, let it stand for 5 minutes, and collect the effluent. Repeat three times; measure the OD280 of the effluent to estimate the antibody concentration. If a large amount of antibody is obtained, SDS-PAGE can be used to detect the purity; add 2 / 5 volume of 1M Tris to the eluted antibody, neutralize at pH 8.0, and switch to the required buffer using a Millipore protein concentrate tube. The buffer is 2×PBS containing 0.02% NaN3 and 1mM... EDTA; concentrate to the required volume to obtain the purified polyclonal antibody against *Bassula marneffei* colp6-2 protein, add an appropriate amount of glycerol, and store at -20°C to avoid freezing.

[0058] Example 2: Preparation of a detection kit for *Basilella marneffei*

[0059] The polyclonal antibody against the *Basilella marneffei* colp6-2 protein prepared in Example 1 was labeled with biotin using conventional techniques to obtain a biotin-labeled polyclonal antibody against the *Basilella marneffei* colp6-2 protein.

[0060] The detection kit for Bassula marneffei includes the following reagents: ELISA microplate, lyophilized standards, dilution buffers for samples and standards, biotin-labeled polyclonal antibody against Bassula marneffei colp6-2 protein, antibody dilution buffer, HRP-streptavidin conjugate (SABC), SABC dilution buffer, TMB substrate solution, stop solution, and concentrated wash buffer.

[0061] The lyophilized standards, dilution buffers for samples and standards, antibody dilution buffers, HRP-streptavidin conjugate (SABC), SABC dilution buffers, TMB substrate solutions, stop solutions, and concentrated wash solutions were all purchased from Wuhan Baiqi Biotechnology Co., Ltd.

[0062] Example 3

[0063] The kit from Example 2 was used to detect whether *Basilella marneffei* was present in the sample.

[0064] 1. Sample collection and storage

[0065] 1.1 Serum: Serum samples should be incubated at room temperature for 2 hours or overnight at 2-8°C, then centrifuged at 1000×g for 20 minutes. The supernatant should be collected and analyzed immediately. Blood collection tubes must be disposable and free of pyrogens and endotoxins.

[0066] 1.2 Plasma: Collect plasma using EDTA-Na2 or heparin as an anticoagulant. Centrifuge at 1000×g for 15 minutes at 2-8℃ within 30 minutes after collection, collect the supernatant and perform analysis immediately. Avoid samples containing hemolysis or high cholesterol.

[0067] 1.3 Tissue Homogenization: Wash tissues with pre-chilled PBS buffer (0.01M, pH=7.4) to remove residual blood. Weigh the tissues, mince them, and homogenize them on ice with PBS buffer (the volume of PBS buffer added depends on the weight of the tissue. Normally, use 9 ml of PBS buffer per 1 gram of tissue fragments. Some protease inhibitors can be added to the PBS buffer). If further cell disruption is required, the suspension can be sonicated using an ultrasonic homogenizer, or the sample can be repeatedly frozen and thawed. Then, centrifuge the homogenate at 5000×g for 5 minutes to obtain the supernatant. Determine the total protein concentration using a BCA kit; the total protein in each well sample should not exceed 0.3 mg.

[0068] 1.4 Cell Culture Supernatant: Centrifuge the culture supernatant at 1000×g for 20 minutes at 2-8℃ to remove insoluble impurities and cell debris. Collect the clear supernatant and perform analysis immediately.

[0069] 1.5 Cell culture lysate: Soluble proteins can be extracted using a commercial RIPA kit, and the total protein concentration can be determined using a BCA kit. The total protein concentration in each well should not exceed 0.3 mg.

[0070] 1.6 Other biological samples: Centrifuge the samples at 1000×g for 20 minutes at 2-8℃. Collect the supernatant and perform the analysis immediately.

[0071] The concentration of the analyte in the sample was assessed through preliminary experiments and by referring to relevant literature, and then diluted appropriately with PBS buffer according to the actual situation.

[0072] 2. Reagent preparation

[0073] 2.1 Washing buffer: Dilute the concentrated washing solution 25 times with deionized water to obtain the washing buffer, and set aside for later use;

[0074] 2.2 Standard Products:

[0075] 2.2.1 Add 1 mL of sample and standard dilution buffer to a test tube containing lyophilized standard, let stand at room temperature for 10 min, then mix thoroughly to obtain sample dilution solution, and label this test tube as zero tube;

[0076] 2.2.2 Take seven more EP tubes and label them 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, and blank, respectively. Add 0.3 ml of sample diluent to each tube. Add 0.3 ml of the standard solution from the blank tube to the first EP tube and mix thoroughly. Transfer 0.3 ml of the mixed standard solution from the first EP tube to the second EP tube and mix thoroughly. Transfer 0.3 ml of the mixed standard solution from the second tube to the third tube and mix thoroughly, and so on. The blank EP tube contains only sample diluent.

[0077] 2.3 Biotin-labeled antibody working solution: according to V 抗体稀释缓冲液 :V 生物素标记的马尔尼菲篮状菌colp6-2蛋白多克隆抗体 The biotin-labeled polyclonal antibody against the colp6-2 protein of *Basilella marneffei* was diluted at a ratio of 99:1 to obtain the working solution of the biotin-labeled antibody.

[0078] 2.4HRP-Streptavidin Coupled Compound (SABC) Working Solution: (Prepared according to V) SABC稀释缓冲液 :V SABC The HRP-streptavidin conjugate (SABC) was diluted at a ratio of 99:1 to obtain the working solution of HRP-streptavidin conjugate (SABC).

[0079] 3. Testing Steps

[0080] 3.1 Rinse the ELISA microplate with washing buffer, and set up standard wells and sample wells on the ELISA microplate;

[0081] 3.2 Sample addition: Add 100 μL of standard or test solution to the corresponding well, cover with a membrane, and incubate at 37°C for 90 min;

[0082] 3.3 Washing: Remove the membrane, wash the plate twice with washing buffer, and after the last wash, remove all washing buffer by aspiration or decanting;

[0083] 3.4 Add biotin-labeled antibody working solution: Add 100 μL of biotin-labeled antibody working solution to each well, cover with a new membrane, and incubate at 37°C for 60 min;

[0084] 3.5 Washing: Remove the membrane and wash the plate 3 times with washing buffer, soaking for 1 minute each time. After the last wash, remove all washing buffer by aspiration or pouring.

[0085] 3.6 Add HRP-Streptavidin Coupled Compound (SABC): Add 100 μL of HRP-Streptavidin Coupled Compound (SABC) working solution to each well, cover with a new membrane, and incubate at 37 °C for 90 min;

[0086] 3.7 Washing: Remove the membrane and wash the plate 3 times with washing buffer, soaking for 1-2 minutes each time. After the last wash, remove all washing buffer by aspiration or pouring.

[0087] 3.8 Add TMB substrate solution: Add 90 μL of TMB substrate solution to each well, cover with a new membrane, and incubate at 37°C for 10-20 min;

[0088] 3.10 Add stop solution: Add 50 μL of stop solution to each well;

[0089] 3.11 Measurement of OD value: After adding the stop solution, immediately measure the absorbance at 450 nm using an ELISA reader and read the OD450 value.

[0090] 3.12 Calculation:

[0091] By measuring the OD450 of standards at various concentrations, a standard curve is plotted. The standard curve is plotted as the relationship between the OD450 value of each gradient of the standard solution (Y-axis) and the corresponding concentration of the standard solution (X-axis).

[0092] By substituting the OD450 value of the sample into the standard curve, it can be determined whether the sample is positive for *Brachysmus marneffei*, and also the target concentration of the sample.

[0093] 4. Recovery rate

[0094] A certain amount of *Bambusa marneffei* colp6-2 protein was incorporated into the sample to be tested, and the detection was performed according to the method described above. The measured value was compared with the expected amount of *Bambusa marneffei* colp6-2 protein in the sample. The results are shown in Table 1.

[0095] Table 1 Recovery Rate

[0096] sample Recovery range (%) Average recovery rate (%) Serum (n=5) 95-104 99 EDTA plasma (n=5) 87-101 95 Heparin plasma (n=5) 86-100 91

[0097] As shown in Table 1, the recovery rate reached 91-99%, indicating that the polyclonal antibody and the detection method have good practicality and accuracy.

[0098] 5. Test Results

[0099] Using the method of this embodiment, samples from 10 patients were tested, and the results are shown in Table 2:

[0100] Table 2 Actual test results

[0101]

[0102] As can be seen from Table 2, all 10 patients carried *Brachys malneffei* to varying degrees. By comparing the OD450 value with the standard curve of the standard, the target concentration in the sample can also be determined.

[0103] Sample 1: Male, 43 years old. Basic information: Positive HIV antibody test for over ten years; current antiviral regimen is "3TC + Kaletra"; history of intravenous drug use for approximately 6 years; previously used methadone for detoxification, now cured. CD4+ T lymphocytes = 234 / µL; Staphylococcus aureus in blood culture. Discharge diagnosis: Bacteremia, chronic renal insufficiency, acquired immunodeficiency syndrome, electrolyte imbalance (hyperkalemia, hyponatremia, hypomagnesemia), grade 3 hypertension, moderate anemia, hyperlipidemia, hyperuricemia, upper respiratory tract infection.

[0104] Sample 2: Female, 56 years old. Basic information: Diagnosed with acquired immunodeficiency syndrome in March 2021, and subsequently received oral antiviral therapy with TDF + 3TC + EFV. Cryptococcus neoformans was found in the cerebrospinal fluid, and the treatment regimen was adjusted to amphotericin B + flucytosine antifungal therapy. CD4+ T lymphocytes = 101 cells / µL.

[0105] Sample 3: Male, 52 years old. Basic information: HIV antibody positive, awaiting follow-up test for 1 week; CD4+ T lymphocytes = 26 cells / µL; carbohydrate antigen = CA15-3170.39U / mL; Mycoplasma pneumoniae IgM antibody = 0.24COI. Cryptococcus neoformans capsular polysaccharide antigen (qualitative) test = negative (-); total fungal glucan content = 761.7pg / ml. Discharge diagnosis: Acquired immunodeficiency syndrome, Pneumocystis pneumonia, bacterial pneumonia, deep fungal infection, likely secondary pulmonary tuberculosis, cytomegalovirus infection, urticaria, liver dysfunction.

[0106] Sample 4: Male, 64 years old. Basic information: He has been undergoing anti-HIV treatment for more than 9 years, and his routine HIV medication collection shows no fungal infection.

[0107] Sample 5: Male, 51 years old. Basic information: In September 2021, he was diagnosed at a hospital in Guangxi Province with HIV antibody undetermined, emaciation, hypoproteinemia, and CD4+ T lymphocytes = 164 cells / µL. Discharge diagnosis: Highly probable acquired immunodeficiency syndrome, HIV wasting syndrome, fungal pneumonia, and mild anemia.

[0108] Sample 6: Female, 39 years old. Discharge diagnosis: Hospitalized at Pingguo People's Hospital in October 2021, diagnosed with "HIV antibody screening positive, malondiform infection of Marneffei, electrolyte imbalance, pulmonary infection, urinary tract infection, moderate anemia, liver dysfunction, hypoalbuminemia, splenomegaly", CD4+ T lymphocytes = 8 / µL. Discharge diagnosis: Acquired immunodeficiency syndrome, malondiform infection of Marneffei, bacterial pneumonia, severe anemia, hypoalbuminemia.

[0109] Sample 7: Male, 28 years old. Basic information: HIV antibody test positive at Guilin CDC in 2018; currently not receiving anti-HIV treatment; CD4+ T lymphocytes = 11 cells / µL; fungal dextran test = 318.6 pg / ml. Discharge diagnosis: Acquired immunodeficiency syndrome, malonfibrillariasis, Pneumocystis pneumonia, herpes simplex, herpetic conjunctivitis.

[0110] Sample 8: Male, 37 years old, on HIV testing for over one month. Diagnosis: AIDS, disseminated penicillinosis, anemia.

[0111] Sample 9: Male, 35 years old. Basic information: CD4+ T lymphocytes = 4 cells / µL, Aspergillus galactomannan content = 5.00µg / L, Candida mannan content = 500.00pg / ml. Discharge diagnosis: Highly probable acquired immunodeficiency syndrome, Pneumocystis pneumonia, oral fungal infection, liver dysfunction, fatty liver, mass in the right supraclavicular fossa, nature to be determined.

[0112] Sample 10: Male, 57 years old. Basic information: Diagnosed with HIV more than 6 years ago, irregular antiretroviral therapy, stopped 3 months ago. History of type 2 diabetes for many years, irregular hypoglycemic therapy. CD4+ T lymphocytes = 4 / µL, Candida mannan content = 500.00 pg / ml, Treponema pallidum specific antibody test = positive (+), sputum Mycobacterium tuberculosis complex DNA = positive (+), blood culture = Bacteroides marneffei. Discharge diagnosis: Acquired immunodeficiency syndrome, secondary pulmonary tuberculosis, intestinal tuberculosis, Bacteroides marneffei infection, pulmonary infection (bacterial + fungal), cytomegalovirus viremia, polyserostomy, type 2 diabetes, lacunar infarction, latent syphilis, drug-induced hepatitis, severe anemia, hypokalemia, hypocalcemia.

[0113] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for preparing a polyclonal antibody against the colp6-2 protein of *Basilella marneffei*, characterized in that, Includes the following steps: Step S1: The antigen sequence colp6-2 is determined by sequence alignment. The amino acid sequence of the antigen sequence colp6-2 is shown in SEQ ID NO.1, and the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO.

2. Step S2: The nucleotide sequence shown in SEQ ID NO.2 is transferred into the expression vector pET28a to construct a recombinant expression vector. The recombinant expression vector is then transformed into Escherichia coli, and the plasmid is extracted. Step S3: Transform the plasmid of the recombinant expression vector into Escherichia coli to obtain a recombinant expression strain, express the target protein in the recombinant expression strain, and purify to obtain the recombinant protein; Step S4: Immunize rabbits with inactivated *Basilus marneffei*, collect blood, centrifuge to obtain antiserum, and purify the antiserum with the recombinant protein obtained in step S3 to obtain a polyclonal antibody against *Basilus marneffei* colp6-2 protein.

2. The preparation method according to claim 1, characterized in that, The method for constructing the recombinant expression vector in step S2 is as follows: using cDNA of *Basilella marneffei* as a template, PCR amplification is performed to obtain the nucleotide sequence shown in SEQ ID NO.

2. This nucleotide sequence is then transferred into the restriction site between BamHI and XhoI of the expression vector pET28a to obtain the recombinant expression vector. The upstream primer sequence used for the PCR amplification is shown in SEQ ID NO.3, and the downstream primer sequence is shown in SEQ ID NO.

4.

3. The preparation method according to claim 1, characterized in that: In step S2, the recombinant expression vector is transformed into Escherichia coli BL21(DE3), and E. coli transformed into the recombinant expression vector are screened using a plate medium containing kanamycin, and then the plasmid is extracted.

4. The preparation method according to claim 1, characterized in that: In step S3, the plasmid of the recombinant expression vector is transformed into Escherichia coli BL21(DE3), and the recombinant expression strain is screened using ampicillin-containing plate medium.

5. The preparation method according to claim 1, characterized in that: In step S3, the recombinant expression strain is inoculated into a liquid culture medium containing ampicillin to obtain a bacterial culture. IPTG inducer is added to induce the expression of the target protein. Then, the bacterial cells are collected by centrifugation, and after ultrasonic disruption, the bacterial cells are purified using a Ni-NTA column to obtain the recombinant protein.

6. The preparation method according to claim 1, characterized in that, In step S4, the method for purifying antiserum with the recombinant protein obtained in step S3 to obtain polyclonal antibodies against the *Bambusa marneffei* colp6-2 protein is as follows: the recombinant protein obtained in step S3 is prepared into an antigen affinity column, the mixture of antiserum and buffer is loaded onto the column, and then eluted with 0.1 M citric acid solution. The eluent is collected, concentrated, and then the polyclonal antibody against the *Bambusa marneffei* colp6-2 protein is obtained.

7. A polyclonal antibody against the colp6-2 protein of *Basilella marneffei* obtained by any of the preparation methods described in claims 1-6.

8. The application of the polyclonal antibody against *Basiliformis marneffei* colp6-2 protein according to claim 7, wherein it is any one of the following (a1) to (a6): (a1) Identification of *Basilella marneffei*; (a2) Prepare a kit for identifying *Basilella marneffei*; (a3) Detect whether the pathogenic microorganism to be tested is *Basilella marneffei*; (a4) Prepare a kit for detecting whether the pathogenic microorganism to be tested is *Basilella marneffei*; (a5) Detect whether the sample to be tested contains *Basilella marneffei*; (a6) Prepare a kit for detecting whether a sample contains *Brachys marneffei*. The application is in non-disease diagnosis and treatment.

9. A reagent kit, characterized in that, Contains the polyclonal antibody against the *Bambusa marneffei* colp6-2 protein as described in claim 7.

10. The application of the kit according to claim 9 is any one of the following (b1) to (b3): (b1) Identification of *Basilella marneffei*; (b2) To determine whether the pathogenic microorganism to be tested is *Basilella marneffei*; (b3) Detect whether the sample to be tested contains *Basilella marneffei*; The application is in non-disease diagnosis and treatment.

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